1. Field of the Invention
The present invention relates to a dry analysis element for the analysis of iron ions in an aqueous liquid sample. Particularly, it relates to a dry analysis element which is suited for the analysis of ferrous ions (Fe.sup.2+) in the presence of cupric ions (Cu.sup.2+) and zinc (Zn.sup.2+).
2. Prior Art
Various methods have been known in the art to analyze the content of iron ions in living body fluids, such as blood, plasma, serum and urine. For example, iron ions in the human serum have been analyzed in the clinical tests for the diagnosis of anemia, liver troubles or lead poisoning.
The atomic adsorption method is the best method for the analysis of the content of iron ions because it has the superior accuracy and sensitivity. However, this method requires a special apparatus and skillfulness and is time consuming method, and thus it is not suited for the practice of analysis in a clinical laboratory or sickroom room of a hospital. The chelatometoric analysis, wherein a chelate compound forming a coloring complex with an iron ion is used, has been widely used as the clinical test.
Various chelating agents have been known for forming complexes with ferrous ions (Fe.sup.2+). Remarkable progress has been made in development of the chelating agents for use in detection of Fe.sup.2+ ions. For instance, although the conventionally known complex of o-phenanthroline or Ferrozine and Fe.sup.2+ ions has a molecular extinction coefficient ranging from 1.times.10.sup.-4 to 3.times.10.sup.-4 mol.sup.-1 cm.sup.-1, complexes of the derivative of pyridylazo compounds developed by Shibata et al. "Bunseki-Kagaku" (Analytical Chemistry), 23, 1412-1430 (1974)) have the molar extinction coefficients of higher than the range of 7.times.10.sup.-4 to 10.times.10.sup.-4 mol.sup.-1 cm.sup.-1 to remarkably improve the sensitivity for the detection of ferrous ions.
With the development of such chelating agents which provide high sensitivity in detection of ferrous ions, it has been tried to analyze ferrous ions in so-called dry methods in lieu of the conventional wet methods, which are simple in construction and operation. In so-called wet method, a reagent is once dissolved in an aqueous medium to prepare a reagent solution, to which a sample to be analyzed is added and a formed coloring product is measured through a colorimeter. On the contrary, in dry method, an aqueous sample is directly spotted on a dry analysis element composed of a test piece, analysis slide or analysis tape containing a reagent composition in a dry state, and the degree of a coloring matter in the dry analysis element is colorimetrically determined. Accordingly, the dry analysis methods are superior over the wet system, since the former provide a result of analysis by a simple operation within a short time.
One example of such a dry analysis element for the analysis of iron ions is disclosed in Unexamined Japanese Patent Publication No. 21368/1989 (corresponding to U.S. Pat. No. 4,789,525 and EP 0 296 795A; meantime throughout this specification, numbers in the parentheses following to Japanese Publication Nos. indicate coresponding foreign Publications).
In dry method for the analysis of ferrous ions, it is required that (1) the molecular extinction coefficient of the formed complex of ferrous ion and chelating agent is high enough for providing a satisfactory sensitivity, that (2) the reaction rate for forming the complex is sufficiently high not to spoil the simplicity and prompt response of dry analysis method, and that (3) the reaction has a high selectivity to exclude the influence by other interfering or hindering ions. In general, chelating agents forming complexes with ferrous ions also form coloring complexes with cupric (Cu.sup.2+) and zinc ions (Zn.sup.2+). The blood serum of a normal human being contains 90 to 180 .mu.g/dl of Fe.sup.2+, 100 to 200 .mu.g/dl of Cu.sup.2+ and 90 to 120 .mu.g/dl Zn.sup.2+, the contents of Cu.sup.2+ and Zn.sup.2+ being comparable to the content of Fe.sup.2+. Accordingly, when the content of ferrous ions in a human blood serum sample is analyzed, the slectivity to ferrous ions against the reactions of these interfering ions is an important factor.
In the prior art dry analysis element disclosed in Unexamined Japanese Patent Publication No. 21368/1989, a pyridylazo compound is used as the chelating agent for forming a chelate complex with iron ions (Fe.sup.2+). The pyridylazo compound used in this prior art dry analysis element is an oil-soluble chelating agent and combined with a coupler solvent (oil) having a high dielectric constant for allowing impregnation with polar ions to exclude interferences by the co-existing Cu.sup.2+ and Zn.sup.2+ ions. However, interferences by these hindering ions cannot be excluded or suppressed to a satisfactorily low level at the pH (4 to 5) condition under the practical use. In order to preclude Cu.sup.2+ ions, a chelating agent, such as neocuproine, is used together. However, the interference by Zn.sup.2+ ions which form a complex having a relatively high molecular extinction coefficient under the practical pH condition cannot be excluded to a satisfactorily low level.
Furthermore, since an oil-soluble chelating agent is used in the prior art technology, there arises a problem that the reaction of the used chelating agent and hydrophilic Fe.sup.2+ ions is retarded.